Optical sensor containing particles for in situ measurement of analytes

ABSTRACT

The invention relates to a sensor for the in vivo measurement of an analyte, comprising a plurality of particles of suitable size such that when implanted in the body of a mammal the particles can be ingested by macrophages and transported away from the site of implantation, each particle containing the components of an assay having a readout which is an optical signal detectable transdermally by external optical means, and either each particles being contained within a biodegradable material preventing ingestion by the macrophages, or each particle being non-biodegradable. The invention relates to a process for the detection of an analyte using such a sensor, comprising implantation of the sensor into the skin of a mammal, transdermal detection of analyte using external optical means, degradation of the biodegradable material, ingestion of the particles by macrophages, and removal of the particles from the site of implantation by macrophages.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the US national phase of international applicationPCT/EP02/07108 filed in English on 27 Jun. 2002, which designated theUS. PCT/EP02/07108 claims priority to GB Application No. 0116853.3 filed10 Jul. 2001. The entire contents of these applications are incorporatedherein by reference.

The present invention relates to a sensor for use in the measurement ormonitoring of analytes in body fluid using optical techniques. Thesensor is particularly suitable for use in situations in which analytelevels must be closely monitored, for example with drugs that must bemaintained within a narrow therapeutic window or where analytemeasurements must be taken repeatedly, such as in diabetes management.

BACKGROUND OF THE INVENTION

In the management of diabetes, the regular measurement of glucose in theblood is essential in order to ensure correct insulin dosing.Furthermore, it has been demonstrated that in the long term care of thediabetic patient better control of the blood glucose levels can delay,if not prevent, the onset of retinopathy, circulatory problems and otherdegenerative diseases often associated with diabetes. Thus there is aneed for reliable and accurate self-monitoring of blood glucose levelsby diabetic patients.

Currently, blood glucose is monitored by diabetic patients with the useof commercially available calorimetric test strips or electrochemicalbiosensors (e.g. enzyme electrodes), both of which require the regularuse of a lancet-type instrument to withdraw a suitable amount of bloodeach time a measurement is made. On average, the majority of diabeticpatients would use such instruments to take a measurement of bloodglucose twice a day. However, the US National Institutes of Healthrecently recommended that blood glucose testing should be carried out atleast four times a day, a recommendation that has been endorsed by theAmerican Diabetes Association. This increase in the frequency of bloodglucose testing imposes a considerable burden on the diabetic patient,both in terms of financial cost and in terms of pain and discomfort,particularly in the long-term diabetic who has to make regular use of alancet to draw blood from the fingertips. Thus, there is clearly a needfor a better long-term glucose monitoring system that does not involvedrawing blood from the patient.

There have been a number of recent proposals for glucose measurementtechniques that do not require blood to be withdrawn from the patient.Various attempts have been made to construct devices in which an enzymeelectrode biosensor is placed on the end of a needle or catheter whichis inserted into a blood vessel (Wilkins, E. and Atanasov, P, Med. Eng.Phys (1996) 18: 273–288). Whilst the sensing device itself is locatedwithin a blood vessel, the needle or catheter retains connection to theexternal environment. In practice, such devices are not suitable for usein human patients first because the insertion of a needle or catheterinto a blood vessel poses an infection risk and is also uncomfortablefor the patient and hence not suitable for continuous use. Secondly,devices of this type have not gained approval for use in patientsbecause it has been suggested that the device itself, on the end of aneedle or catheter, may be responsible for the shedding of thrombosesinto the patient's circulation. This obviously poses a very serious riskto the patient's health.

Mansouri and Schultz (Biotechnology 1984), Meadows and Schultz (Anal.Chim. Acta. (1993) 280: pp 21–30) and U.S. Pat. No. 4,344,438 alldescribe devices for the in situ monitoring of low molecular weightcompounds in the blood by optical means. These devices are designed tobe inserted into a blood vessel or placed subcutaneously but requirefibre-optic connection to an external light source and an externaldetector. Again the location of these devices in a blood vessel carriesan associated risk of promoting thromboses and in addition, in oneembodiment the need to retain a fibre-optic connection to the externalenvironment is impractical for long-term use and carries a risk ofinfection.

In the search for a less invasive glucose monitoring technique someattention has also been focussed on the use of infra-red spectroscopy todirectly measure blood glucose concentration in blood vessels in tissuessuch as the ear lobe or finger tip which are relatively “lighttransparent” and have blood vessels sited close to the surface of theskin (Jaremko, J. and Rorstad, O. Diabetes Care 1998 21: 444–450 andFogt, E. J. Clin. Chem. (1990) 36: 1573–80). This approach is obviouslyminimally invasive, but has proven to be of little practical value dueto the fact that the infra-red spectrum of glucose in blood is sosimilar to that of the surrounding tissue that in practical terms it isvirtually impossible to resolve the two spectra.

It has been observed that the concentration of analytes in subcutaneousfluid correlates with the concentration of said analytes in the blood,and consequently there have been several reports of the use of glucosemonitoring devices which are sited in a subcutaneous location. Inparticular, Atanasov et al. (Med. Eng. Phys. (1996) 18: pp 632–640)describe the use of an implantable glucose sensing device (dimensions5.0×7.0×1.5 cm) to monitor glucose in the subcutaneous fluid of a dog.The device consists of an amperometric glucose sensor, a miniaturepotentiostat, an FM signal transmitter and a power supply and can beinterrogated remotely, via antenna and receiver linked to acomputer-based data acquisition system, with no need for a connection tothe external environment. However, the large dimensions of this devicewould obviously make it impractical for use in a human patient.

Ryan J. Russell et al, Analytical Chemistry, Vol. 71, Number 15,3126–3132 describes an implantable hydrogel based on polyethyleneglycolcontaining fluorescein isothiocyanate dextran (FITC-dextran) andtetramethylrhodamine isothiocyanate concavalin A chemically conjugatedto the hydrogel network for dermal implantation. The implanted hydrogelspheres are to be transdermally interrogated.

R. Ballerstadt et al, Analytica Chemica Acta, 345 (1997), 203–212discloses an assay system in which two polymer (dextran) molecules arerespectively labelled with first and second fluorophores and are boundtogether by multivalent lectin molecules, producing quenching. Glucosesaturates the binding sites of the lectin, causing disassociation of thetwo polymers, giving an increase in fluorescence.

Joseph R. Lakowicz et al, Analytica Chimica Acta, 271, (1993), 155–164describes the use of phase modulation fluorimetry. This substitutes afluorescence lifetime based measurement for the fluorescence intensitybased measurements taught in the earlier described art.

Fluorescence lifetime can be measured by a phase modulation technique byexciting fluorescence using light which is intensity modulated at 1 to200 MHz and measuring the phase shift of the emission relative to theincident light and the modulation of the emission.

In WO91/09312 a subcutaneous method and device is described that employsan affinity assay for glucose that is interrogated remotely by opticalmeans. In WO97/19188 a further example of an implantable assay systemfor glucose is described which produces an optical signal that can beread remotely. The devices described in WO91/09312 and WO97/19188 willpersist in the body for extended periods after the assay chemistry hasfailed to operate correctly and this is a major disadvantage for chronicapplications. Removal of the devices will require a surgical procedure.

WO00/02048 deals with this problem by using a biodegradable material tocontain the assay reagents. There the assay materials would be likely tobe in contact with the bloodstream once the biodegradable material hasdegraded. It would be desirable to minimise or avoid this.

There remains a clear need for sensitive and accurate blood glucosemonitoring techniques which do not require the regular withdrawal ofblood from the patient, which do not carry a risk of infection ordiscomfort and which do not suffer from the practical disadvantages ofthe previously described implantable devices.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood with reference to thefollowing non-limiting examples, together with the accompanying figuresin which:

FIG. 1 is a schematic diagram of the optical part of the fibre opticfluorimeter; and

FIG. 2 is a schematic diagram of a driver/amplifier circuit used inconjunction with the optical part of the fibre optic fluorimeter.

DESCRIPTION OF THE INVENTION

Accordingly, in a first aspect the present invention provides a sensorfor the in vivo measurement of an analyte, comprising a plurality ofparticles of suitable size such that when implanted in the body of amammal the particles can be ingested by macrophages and transported awayfrom the site of implantation, each particle containing the componentsof an assay having a readout which is an optical signal detectable ormeasurable transdermally by external optical means, and either eachparticle being contained within a biodegradable material preventingingestion by macrophages, or each particle being non-biodegradable.

The sensor particles are preferably embedded within a matrix ofbiodegradable material, but may alternatively be retained by an envelopeof biodegradable material, or may be separately covered withbiodegradable material.

Preferably, the particles are less than 5 μm in their largest dimension.

The sensor may be introduced within the skin by injection, preferablyusing a syringe, or by other methods, in particular by any methodsdescribed in WO00/02048. The sensor may be introduced within thethickness of the dermis, or subdermally, or may be introduced to theepidermis, although in the latter case it would be likely to be expelledfrom the skin by outgrowth of the epidermal layers, possibly before thebiodegradable material, if present, has degraded.

Because the sensor is located within the skin, an optical signalgenerated in the sensor particles can be detected transcutaneously (i.e.through the higher layer(s) of the skin) thus obviating the need for anydirect connection between the sensor and the external environment. Oncethe sensor is in place in a cutaneous location analyte measurements canbe taken as often as is necessary with no adverse effects. This is aparticular advantage in relation to the long-term care of diabeticpatients because if glucose measurements are taken more frequently,tighter control can be maintained over the level of glucose in the bloodand the risk of developing conditions related to poorly regulated bloodglucose, such as retinopathy, nephropathy, neuropathy, general micro-and macrovascular damage and poor circulation, will be reduced.

The biodegradable material if present and sensor particles arepreferably permeable to body fluid, thereby allowing analytes such asglucose to enter the particles by diffusion and to interact with thecomponents of the assay.

Because the sensor of the invention does not itself contain any of theoptical components required to interrogate the readout of the assay(these being provided separately and located outside the body) thesensor can easily be provided in a form which is injectable with minimaldiscomfort to the patient.

The biodegradable material may be an injectable formulation that forms agel at the point of injection within the skin of the patient. The sensorparticles may be formed from a solid polymeric material incorporatingthe components of the assay which is again injected or implantedcutaneously, the polymeric material typically being of a size suitablefor injection through a narrow gauge needle to minimise the discomfortto the patient. When placed cutaneously the solid polymeric materialabsorbs water and expands to form a gel, thus hydrating the componentsof the assay.

The biodegradable material may contain the sensor particles such thatthey are held in position at the site of injection. This enables opticalmeasurements to be taken at the skin surface over this position. Theassay contained in all sensor particles may thus be interrogatedsimultaneously, to give a measureable signal.

The biodegradable material, if present, will degrade over a period oftime within the skin, releasing the sensor particles. At this point theuseful lifetime of the sensor is over, and it is desirable to remove theparticles from the body or to contain the particles within the body awayfrom the sensing site. If the particles are to be removed from the body,it is desirable to achieve this non-surgically.

Macrophage cells occur in the body as part of the immune system. Thesecells are produced in the bone marrow, and are capable of ingestingforeign particles (including necrotic cells) by surrounding suchparticles with extrusions of the cell membrane in a process calledphagocytosis. Macrophages also secrete enzymes which damage foreignorganisms.

Macrophages are able to ingest and transport particles up to a certainsize, e.g. 5 μm in largest dimension. Therefore, when the sensorbiodegradable material of this invention degrades to release suitablysmall sensor particles, the sensor particles will be ingested bymacrophages. The sensor particles may also be ingested by othercomponents of the immune system.

Alternatively, if the sensor particles are not contained within abiodegradable material, the sensor particles will be ingested bymacrophages without a delay for degradation of the biodegradablematerial. Optical measurements must thus be taken in the relativelyshort period between introduction of the sensor particles to the bodyand removal of the sensor particles by macrophages.

Preferably, the sensor particles of the invention have suitable surfacecharacteristics for ingestion by macrophages.

Following phagocytosis, macrophages travel to the lymphatic system. Ifthe sensor particles are biodegradable, their components will be removedby the lymphatic system. If the sensor particles are not biodegradable,they will remain in the lymph nodes. This means that the reagents of theinvention will be eliminated via the lymphatic system rather than beingreleased in the skin.

The particles contained within biodegradable material of the presentinvention may be biodegradable or hydrolysable in vivo, such that theymay be digested by macrophages, but this is not necessary. It isundesirable that the particles degrade before they are ingested bymacrophages, since this may expose the bloodstream to the assay reagentmaterials.

Each of the sensor particles may contain the assay components eitherencapsulated inside a hollow microparticle, or dispersed within thematerial of a solid microparticle. Techniques for forming suchmicroparticles are known in the art. Typically, assay components,polymer and solvent are combined to form a droplet. The solvent isremoved and the droplets are collected, dried and filtered to product adry, free-flowing powder of solid microparticles containing dispersedassay components. Alternatively, emulsion or coacervation techniques maybe used. Both processes may incorporate stabilisation techniques.

Alternatively, for particles contained within biodegradable material,liposomes containing the assay components can be used. In a furtherembodiment, each sensor particle comprises an empty erythrocyte whichhas been loaded with assay components. Empty erythrocytes, also known aserythrocyte ghosts, can be prepared by exposing intact erythrocytes to ahypotonic solution so that they swell and burst to release theircytoplasmic contents. The empty erythrocytes can then be loaded withassay components before allowing the plasma membranes to reseal.

Materials suitable as biodegradable materials of a sensor of theinvention include biodegradable block copolymers such as those describedby Jeong et al., Nature 388: pp 860–862. Aqueous solutions of thesematerials are thermosensitive, exhibiting temperature-dependentreversible gel-sol transitions. The polymer biodegradable material canbe loaded with the sensor particles at an elevated temperature where thematerial forms a sol. In this form the material is injectable and oncutaneous injection and subsequent rapid cooling to body temperature thematerial forms a gel matrix. The sensor particles are suspended withinthis gel matrix which thus constitutes a sensor suitable for detectingor measuring analytes in body fluid. Low molecular weight analytes, suchas glucose, can freely diffuse into the gel matrix from the surroundingbody fluid. Cutaneous injection of the sol phase material causes neithersignificant pain or tissue damage.

As an alternative to the gel based sensor described above the sensor maycomprise a solid or gel-like polymer biodegradable material within whichthe sensor particles are distributed. When injected or implantedcutaneously this solid polymer sensor hydrates and swells, and analytepenetrates through the structure to encounter the sensor particles.

Biodegradable materials suitable for use in the construction of thesensors include cross-linked proteins such as human albumin, fibringels, polysaccharides such as starch or agarose, polylactides (PLA) suchas poly (DL-lactide), polyglycolides (PGA) such as poly (DL-glycolide),poly(lactide-co-glycolides) (PLGA), polyanhydrides, polyorthoesters,fatty acid/cholesterol mixtures that form semi-solid derivates,hyaluronates and liquid crystals of monoolein and water. These materialshave the advantage that they are broken down into biologicallyacceptable molecules which are metabolised and removed from the body vianormal pathways.

In the preferred embodiments of the sensor, it is advantageous for theassay components to have a restricted diffusion in order to minimisetheir loss from the sensor particles into the biodegradable material andpotentially into the bloodstream. This can be achieved by ensuring thatthe sensor particles have a pore size that permits the diffusion of lowmolecular weight analytes such as glucose, but not diffusion of theassay components themselves. The assay components are preferably of highmolecular weight, such as proteins or polymers, in order to restricttheir loss from the sensor particles.

Assays suitable for use in the sensor include reactions such ashydrolysis and oxidation leading to detectable optical change i.e.fluorescence enhancement or quenching which can be observedtranscutaneously. A preferred assay for use in the sensor of theinvention is a binding assay, the readout of which is a detectable ormeasurable optical signal which can be interrogated transcutaneouslyusing optical means. The binding assay generating the optical signalshould preferably be reversible such that a continuous monitoring offluctuating levels of the analyte can be achieved. This reversibility isa particular advantage of the use of a binding assay format in which thecomponents of the assay are not consumed. Binding assays are alsopreferred for use in the sensor of the invention for reasons of safetyas they cannot generate any unwanted products as might be generated byan enzymatic or electrochemical reaction.

Preferred binding assay configurations for use in the sensor of theinvention include a reversible competitive, reagent limited, bindingassay, the components of which include an analyte analog and an analytebinding agent capable of reversibly binding both the analyte of interestand the analyte analog. The analyte of interest and the analyte analogcompete for binding to the same binding site on the analyte bindingagent. Such competitive binding assay configurations are well known inthe art of clinical diagnostics and are described, by way of example, inThe Immunoassay Handbook, ed. David Wild, Macmillan Press 1994. Suitableanalyte binding agents for use in the assay would include antibodies orantibody fragments which retain an analyte binding site (e.g. Fabfragments), lectins (e.g. concanavalin A), hormone receptors, drugreceptors, aptamers and molecularly-imprinted polymers. Preferably, theanalyte analog should be a substance of higher molecular weight than theanalyte such that it cannot freely diffuse out of the sensor particles.For example, an assay for glucose might employ a high molecular weightglucose polymer such as dextran as the analyte analog.

Suitable optical signals which can be used as an assay readout inaccordance with the invention include any optical signal which can begenerated by a proximity assay, such as those generated by fluorescenceresonance energy transfer, fluorescence polarisation, fluorescencequenching, phosphorescence technique, luminescence enhancement,luminescence quenching, diffraction or plasmon resonance, all of whichare known per se in the art.

The most preferred embodiment of the sensor of the inventionincorporates a competitive, reagent limited binding assay whichgenerates an optical readout using the technique of fluorescenceresonance energy transfer. In this assay format, the analyte analog islabelled with a first chromophore and the analyte binding agent islabelled with a second chromophore. One of the first and secondchromophores acts as a donor chromophore and the other acts as anacceptor chromophore. It is an essential feature of the assay that thefluorescence emission spectrum of the donor chromophore overlaps withthe absorption spectrum of the acceptor chromophore, such that when thedonor and acceptor chromophores are brought into close proximity by thebinding agent a proportion of the energy which normally would producefluorescence emitted by the donor chromophore (following irradiationwith incident radiation of a wavelength absorbed by the donorchromophore) will be non radiatively transferred to the adjacentacceptor chromophore, a process known in the art as fluorescenceresonance energy transfer, with the result that a proportion of thefluorescent signal emitted by the donor chromophore is quenched, thatthe lifetime of the fluorescence is changed, and, in some instances,that the acceptor chromophore emits fluorescence. The acceptorchromophore may, however, be a non-fluorescent dye. Fluorescenceresonance energy transfer will only occur when the donor and acceptorchromophores are brought into close proximity by the binding of analyteanalog to analyte binding agent. Thus, in the presence of analyte, whichcompetes with the analyte analog for binding to the analyte bindingagent, the amount of quenching is reduced (resulting in a measurableincrease in the intensity of the fluorescent signal emitted by the donorchromophore or a fall in the intensity of the signal emitted by theacceptor chromophore) as labelled analyte analog is displaced frombinding to the analyte binding agent. The intensity or lifetime of thefluorescent signal emitted from the donor chromophore thus correlateswith the concentration of analyte in the subcutaneous fluid bathing thesensor.

An additional advantageous feature of the fluorescence resonance energytransfer assay format arises from the fact that any fluorescent signalemitted by the acceptor chromophore following excitation with a beam ofincident radiation at a wavelength within the absorption spectrum of theacceptor chromophore is unaffected by the fluorescence resonance energytransfer process. It is therefore possible to use the intensity of thefluorescent signal emitted by the acceptor chromophore as an internalreference signal, for example in continuous calibration of the sensor orto monitor the extent to which the sensor has degraded and thus indicatethe need to implant or inject a fresh sensor. As the sensorbiodegradable material degrades and sensor particles are released, theamount of acceptor chromophore present in the sensor will decrease, andhence the intensity of fluorescent signal detected upon excitation ofthe acceptor chromophore will also decrease. The fall of this signalbelow an acceptable baseline level would indicate the need to implant orinject a fresh sensor. Competitive binding assays using the fluorescenceresonance energy transfer technique which are capable of being adaptedfor use in the sensor of the invention are known in the art. U.S. Pat.No. 3,996,345 describes immunoassays employing antibodies andfluorescence resonance energy transfer between a fluorescer-quencherchromophoric pair. Meadows and Schultz (Anal. Chim. Acta (1993 280: pp21–30) describe a homogeneous assay method for the measurement ofglucose based on fluorescence resonance energy transfer between alabelled glucose analog (FITC labelled dextran) and a labelled glucosebinding agent (rhodamine labelled concanavalin A). In all of theseconfigurations the acceptor and donor chromophores/quenchers can belinked to either the binding agent or the analyte analog.

The various FRET chemistries described in the background art cited inthe introduction of this document may be used.

Fluorescence lifetime or fluorescence intensity measurements may bemade. As described in Lakowitz et al., fluorescence lifetime may bemeasured by phase modulation techniques.

An alternative to the fluorescence resonance energy transfer is thefluorescence quenching technique. In this case a compound withfluorescence quenching capability is used instead of the specificacceptor chromophore and the optical signal in a competitive bindingassay will increase with increasing analyte. An example of a powerfuland non-specific fluorescence quencher is given by Tyagi et al. NatureBiotechnology (1998) 18: p 49.

The sensor of the invention can be adapted for the detection orquantitative measurement of any analyte present in body fluid. Preferredanalytes include glucose (in connection with the long-term monitoring ofdiabetics), urea (in connection with kidney disease or dysfunction),lactate (in connection with assessment of muscle performance in sportsmedicine), ions such as sodium, calcium or potassium and therapeuticdrugs whose concentration in the blood must be closely monitored, suchas, for example, digoxin, theophylline or immunosuppressant drugs. Theabove analytes are listed by way of example only and it is to beunderstood that the precise nature of the analyte to be measured is notmaterial to the invention.

The sensor is interrogated transcutaneously using optical means i.e. nophysical connection is required between the sensor and the opticalmeans. When the sensor incorporates a competitive, reagent limited,binding assay employing the technique of fluorescent energy transfer,the optical means should supply a first beam of incident radiation at awavelength within the absorption spectrum of the donor chromophore andpreferably a second beam of incident radiation at a wavelength withinthe adsorption spectrum of the acceptor chromophore. In addition, theoptical means should preferably be capable of measuring optical signalsgenerated in the sensor at two different wavelengths; wavelength 1within the emission spectrum of the donor chromophore (the signalgenerated in connection with the measurement of analyte and wavelength 2in the emission spectrum of the acceptor chromophore (which could be theanalyte signal or the internal reference or calibration signal).

Optical means suitable for use in remote interrogation of the device ofthe invention include a simple high-throughput fluorimeter comprising anexcitation light source such as, for example, a light-emitting diode(blue, green or red), an excitation light filter (dichroic or dyefilter) and a fluorescent light detector (PIN diode configuration). Afluorimeter with these characteristics may exhibit a sensitivity ofbetween picomolar to femtomolar fluorophore concentration.

A suitable fluorimeter set-up is shown in the accompanying FIG. 1 anddescribed in the Examples included herein. The fluorimeter separatelymeasures the following parameters:

At wavelength 1 (donor chromophore)

-   -   Excitation light intensity, I(1,0)    -   Ambient light intensity, I(1,1)    -   Intensity of combined fluorescent and    -   ambient light, I(1,2)

At wavelength 2 (acceptor chromophore)

-   -   Excitation light intensity, I(2,0)    -   Ambient light intensity, I(2,1)    -   Intensity of combined fluorescent and    -   ambient light, I(2,2)

Measurements are taken by holding the fluorimeter close to the skin andin alignment with the sensor. When making transcutaneous measurements ofthe fluorescent signals generated in the sensor it is necessary to takeaccount of the absorption of signal by the skin, the absorptivity ofhuman skin is found by experiment to be lowest in the range from 400 nmto 900 nm. The final output provided is the normalised ratio between thefluorescent intensity from the two fluorophores, defined by thefollowing relation (Equation 1)Final output=(I(1,2)−I(1,1))*I(2,0)/(I(2,2)−I(2,1))*I(1,0)  (1)

The final output from the optical means (e.g. the fluorimeter) as givenby Equation 1 above is converted to analyte concentration preferably bymeans of a computer using calibration data which can be obtained basedon the principles set out below.

A calibration curve can be established empirically by measuring responseversus analyte concentration for a physiologically relevant range ofanalyte concentrations. Preferably, this takes place in vitro as part ofthe production of the sensor device. The calibration procedure can besimplified considerably by using the mathematical relation betweenresponse and analyte concentration in a competitive affinity sensorwhich is derived as follows:

The response of a competitive affinity sensor is governed by thereactions:RC

R+CRL

R+L

Designating the dissociation of the complexes RC and RL, formed by thecombination of analyte binding agent (R) with analyte (L) or analyteanalog (C).

The corresponding dissociation equilibrium constants are:

$K_{1} = \frac{C_{r}C_{c}}{C_{RC}}$${and},\text{}{K_{2} = \frac{C_{r}C_{c}}{C_{RL}}}$where C designates the number of moles of the species in the sensordivided by the sensor volume. Using this measure of concentration bothimmobilised species and species in solution are treated alike.

The mass balance equations are:T _(C) =C _(C) +C _(RC)for total analyte analog concentration and,T _(R) =C _(R) +C _(RC) +C _(RL)for total analyte binding agent concentration.

Using the expression above, the relation between response and analyteconcentration is derived:

$\begin{matrix}{{\frac{T_{C} - C_{C}}{C_{C}}K_{1}} = \frac{T_{R} - \left( {T_{C} - C_{C}} \right)}{1 + \left( {C_{L}/K_{2}} \right)}} & (2)\end{matrix}$By using this relation the amount of data necessary for the calibrationcan be reduced to two key parameters: Total analyte binding agentconcentration and total analyte analog concentration. The calibrationcurve is thus determined by two points on the curve.

In a second aspect, the present invention relates to a process for thedetection of an analyte using a sensor as described herein, comprisingimplantation of the sensor into the skin of a mammal, transdermaldetection or measurement of analyte using external optical means,degradation of the biodegradable material, ingestion of the particles bymacrophages, and removal of the particles from the site of implantationby macrophages.

EXAMPLE 1

A glucose assay according to Meadows and Schultz (Talanta, 35, 145–150,1988) was developed using concanavalin A-rhodamine and dextran-FITC(both from Molecular Probes Inc., Oregan, USA). The principle of theassay is fluorescence resonance energy transfer between the twofluorophores when they are in close proximity; in the presence ofglucose the resonance energy transfer is inhibited and the fluorescentsignal from FITC (fluorescein) increases. Thus increasing fluorescencecorrelates with increasing glucose. The glucose assay was found torespond to glucose, as reported by Schultz, with approximately 50percent recovery of the fluorescein fluorescence signal at 20 mg/dLglucose. Fluorescence was measured in a Perkin Elmer fluorimeter,adapted for flow-through measurement using a sipping device.

EXAMPLE 2

The sensor particles are produced by combining concanavalin A-rhodamineand dextran-FITC with polymer and solvent to form a droplet. The solventis removed, and the droplets are collected, dried and filtered to give adry, free-flowing powder.

The sensor particles are combined with biodegradable material in aninjectable formulation to form the sensor.

EXAMPLE 3

Malachite Green (MG)-Dextran is prepared using the method described inJoseph R. Lakowicz et al., Analytica Chimica Acta, 271 (1993) 155–164.Amino dextran (10 000 MW) is dissolved in pH 9.0 bicarbonate buffer andreacted with an 10-fold excess of MG-isothiocyanate for 4 h at roomtemperature. The labelled dextran is freed from excess fluorophore on aSephadex G-50 column.

Cascade Blue Concanavalin A (Cascade Blue-Con A) is obtained from Sigma.

The sensor particles are produced by combining Cascade Blue-ConcanavalinA and Malachite Green-Dextran with polymer and solvent to form adroplet. The solvent is removed, and the droplets are collected, driedand filtered to give a dry, free-flowing powder.

The sensor particles are combined with biodegradable material in aninjectable formulation to form the sensor.

EXAMPLE 4

A fibre optic fluorimeter was assembled as follows:

The optical part of a fibre optic fluorimeter was made from standardcomponents on a micro bench. The set-up, comprising a red LED as lightsource, lenses, dichroic beamsplitter and filters and detector diodes,was as shown in FIG. 1. Briefly, the fluorimeter comprises a lightemitting diode (1) providing an excitation light beam which passesthrough a condenser (2) containing an excitation filer (3) and isincident upon a beamsplitter (4). Part of the excitatory beam is therebydeflected into launching optics (5) and enters an optical fibre (6).When the fluorimeter is in use in the interrogation of a cutaneouslylocated sensor the end of the skin, in alignment with the cutaneoussensor, so that beam of excitatory light is incident upon the sensor aportion of the optical signal emitted from the sensor followingexcitation enters the optical fibre (6) and is thereby conveyed into thefluorimeter where it passes through a blocking diode (7). Thefluorimeter also contains a reference detector diode (9) which providesa reference measurement of the excitatory light emitted from the LED(1). The ends of a 1 m long Ensign Beckford optical fibre, 0.5 mm indiameter, numerical aperture of 0.65, were ground to a mirror finishusing diamond paste on glass paste. One end of the fibre was mounted inan X Y Z holder in front of a 20× microscope objective. The diodes (LED(1) and detector diodes (7) and (9)) were connected to a custom madedriver/amplifier circuit as shown in FIG. 2. The circuit comprises asender (10), current amplifiers (11) and (12), multiplexers (13) and(14), integrators (15) and (16) and analog divider (17). The drivercircuit was set to drive the LED (1) at 238 Hz and the signals from thedetector diodes (7) and (9) were switched between ground and the storagecapacitors (integrator with a time constant of 1 second) synchronisedwith the drive signal. The two integrated signals correspond tobackground-corrected fluorescent signal and background correctedexcitation light level (LED intensity). The former divided by the latterwas supported by an analog divider as shown in FIG. 2. For testpurposes, the distal end of the fibre (6) was dipped into dilutesolutions of rhodamine and the optics were adjusted for maximum signalfrom the analog divider.

The fluorimeter is battery operated (typical power consumption 150 mA at9 V) and for convenience can be constructed in the shape and dimensionsof a pen.

EXAMPLE 5

The sensor prepared in Example 2 is injected by syringe in the back ofthe hand of a human volunteer.

A fibre optic fluorimeter (see Example 4) is directed at the skin and arhodamine fluorescence lifetime signal is obtained and correlated with aconventional blood glucose measurement indicating that transdermalmeasurements can be made on implanted sensors.

1. A sensor for in vivo measurement of an analyte, comprising aplurality of particles having a size such that when implanted in a bodyof a mammal at an implantation site the particles can be ingested bymacrophages and transported away from the implantation site, whereineach particle of the sensor contains the components of an assay having areadout which is an optical signal detectable transdermally by externaloptical means, and wherein the particles are each contained within abiodegradable material preventing ingestion by the macrophages.
 2. Asensor as claimed in claim 1, wherein the particles are less than 5 μmin their largest dimension.
 3. A sensor as claimed in claim 1, whereinthe assay is a binding assay.
 4. A sensor as claimed in claim 3, whereinthe binding assay is a competitive binding assay, the components ofwhich include an analyte binding agent and an analyte analogue.
 5. Asensor as claimed in claim 4, wherein the analyte analogue is labelledwith a first chromophore and the analyte binding agent is labelled witha second chromophore, an emission spectrum of the first chromophore orthe second chromophore overlapping with an absorption spectrum of thesecond chromophore or the first chromophore respectively.
 6. A sensor asclaimed in claim 4, wherein the binding agent is an antibody, a Fabfragment, a lectin, a hormone receptor, a drug receptor, an aptamer or amolecularly-imprinted polymer.
 7. A sensor as claimed in claim 1,wherein the optical signal is generated by fluorescence resonance energytransfer, fluorescence polarisation, fluorescence quenching,phosphorescence, luminescence enhancement, luminescence quenching,diffraction or plasmon resonance.
 8. A sensor as claimed in claim 1,wherein the analyte is glucose.
 9. A sensor as claimed in claim 1,wherein the optical signal is quantitatively measurable.
 10. A processfor the detection of an analyte using a sensor as claimed in claim 1,comprising the steps of: implanting the sensor into skin of a mammal atan implantation site, and transdermally detecting analyte using externaloptical means, where implantation is followed by degradation of thebiodegradable material, ingestion of the particles by macrophages, andremoval of the particles from the implantation site.
 11. A sensor for invivo measurement of an analyte, comprising a plurality of particleshaving a size such that when implanted in a body of a mammal at animplantation site the particles can be ingested by macrophages andtransported away from the implantation site, wherein each particle ofthe sensor contains the components of an assay having a readout which isan optical signal detectable transdermally by external optical means,and wherein the particles are each contained within a biodegradablematerial preventing ingestion by the macrophages, the biodegradablematerial being a gel.